The subject of the present invention is an adenoviral fiber mutated in the regions involved in the recognition and the binding of the natural cell receptor for adenoviruses. It also relates to the recombinant viruses carrying at their surface such a fiber and a ligand which confers on them a modified or targeted host specificity towards a particular cell type, the cells containing these adenoviruses as well as a method for preparing infectious viral particles thereof intended for therapeutic use. The invention is most particularly of interest for gene therapy perspectives, in particular in humans.
By virtue of their particular properties, adenoviruses are used in an increasing number of applications in gene therapy. Having been identified in numerous animal species, they are not very pathogenic, are nonintegrative and replicate both in dividing and quiescent cells. Furthermore, they exhibit a broad host spectrum and are capable of infecting a very large number of cell types such as epithelial cells, endothelial cells, myocytes, hepatocytes, nerve cells and synoviocytes (Bramson et al., 1995, Curr. Op. Biotech. 6, 590-595). However, this absence of specificity of infection could constitute a limit to the use of recombinant adenoviruses, on the one hand, from a safety point of view since there may be dissemination of the recombinant gene in the host organism and, on the other hand, from the efficiency point of view since the virus does not infect specifically the cell type which it is desired to treat.
In general, the adenoviral genome consists of a double-stranded linear DNA molecule of about 36 kb containing the genes encoding the viral proteins and, at its ends two inverted repeats (designated ITR for Inverted Terminal Repeat) involved in the replication and the encapsidation region. The early genes are distributed in 4 regions dispersed in the adenoviral genome (E1 to E4; E for early), containing 6 transcriptional units equipped with their own promoters. The late genes (L1 to L5; L for late) partly cover the early transcription units and are, for the most part, transcribed from the major late promoter MLP.
As a guide, all the adenoviruses used in gene therapy protocols are deficient for replication by deletion of at least the E1 region and are propagated in a complementation cell line which provides in trans the deleted viral functions. The 293 line, established from human embryonic kidney cells, which efficiently complements the E1 function (Graham et al., 1977, J.
Gen. Virol. 36, 59-72), is commonly used. Second-generation vectors have recently been proposed in the literature. They conserve the regions in cis which are necessary for the replication of the virus in the infected cell (ITRs and encapsidation sequences) and contain substantial internal deletions designed to eliminate most of the viral genes whose expression in vivo can lead to the establishment of inflammatory or immune responses in the host. The adenoviral vectors and the technique for their preparation have been the subject of numerous publications which are accessible to persons skilled in the art.
The infectious cycle for adenoviruses occurs in 2 steps. The early phase precedes the initiation of replication and makes it possible to produce the early proteins regulating the replication and transcription of the viral DNA. The replication of the genome is followed by the late phase during which the structural proteins which constitute the viral particles are synthesized. The assembly of the new virions takes place in the nucleus. In a first stage, the viral proteins assemble so as to form empty capsids of icosahedral structure into which the genome is encapsidated. The adenoviruses liberated are capable of infecting other permissive cells. In this regard, the fiber and the penton base present at the surface of the capsids play a critical role in the cellular attachment of the virions and their internalization.
The adenovirus binds to a cellular receptor present at the surface of the permissive cells via the trimeric fiber (Philipson et al., 1968, J. Virol. 2, 1064-1075; Defer et al., 1990, J. Virol. 64, 3661-3673). The particle is then internalized by endocytosis through the binding of the penton base to the cellular integrins xcex1vxcex23 and xcex1vxcex25 (Mathias et al., 1994, J. Virol. 68, 6811-6814). The capacity of the soluble fiber or of anti-fiber antibodies to inhibit infection demonstrates its role in the cellular attachment of the virus.
The fiber is composed of 3 domains (Chroboczek et al., 1995, Current Top. Microbiol. Immunol. 199, 165-200):
(1) At the N-terminus, the tail, which is highly conserved from one serotype to another, interacts with the penton base and ensures the anchorage of the molecule in the capsid.
(2) The stem is a structure in the form of a rod, composed of a number of repeats of xcex2 sheets, this number varying depending on the serotypes.
(3) Finally, at the distal end of the stem, the head is a spherical globular structure which contains the trimerization signals (Hong and Engler, 1996, J. Virol. 70, 7071-7078; Novelli and Boulanger, 1991, J. Biol. Chem. 266, 9299-9303; Novelli and Boulanger, 1991, Virology 185, 365-376). Furthermore, most of the experimental data show that the head domain is responsible for the binding to permissive cells (Henry et al., 1994, J. Virol. 68, 5239-5246; Louis et al., 1994, J. Virol. 68, 4104-4106).
xe2x80x9cTargetedxe2x80x9d adenoviruses whose native fiber is modified so as to recognize a different cellular receptor have already been proposed in the literature. Thus, WO94/10323 describes mutants of the fiber of Ad5, into which a sequence encoding an antibody fragment (of the scFv type) is inserted at the end of one of the 22 repetitive units of the stem with the aim of modifying the specificity of infection towards cells having the target antigen. U.S. Pat. No. 5,543,328 describes an Ad5 chimeric fiber in which the head domain is replaced by tumor necrosis factor (TNF) so as to interact with the cellular receptor for TNF. In another construct, the Ad5 native fiber is fused at its C-terminal end with the peptide ApoE allowing binding to the LDL (for low density lipoprotein) receptor present at the surface of hepatic cells. WO95/26412 describes a fiber modified by incorporation of a ligand at the C-terminal end which conserves its trimerization capacities. WO96/26281 describes a chimeric fiber obtained by replacing part of the native fiber and, in particular, the head, with the equivalent part of an adenoviral fiber of another serotype and, optionally, by inserting at the C-terminal end a peptide RGD which is specific for vitronectin.
As indicated above, the specificity of infection of an adenovirus is determined by the attachment of the adenoviral fiber to a cellular receptor situated at the surface of permissive cells.
French patent application 97 01005 has identified the role of the antigens of the class I major histocompatibility complex and of the III modules of fibronectin as primary receptor and as cofactor, respectively, for adenoviruses. However, other proteins may be involved. In this regard, recent studies have presumed the use of the cellular receptor for the coxsackie viruses by the types 2 and 5 adenoviruses to penetrate into their target cells (Bergelson et al., 1997, Science 275, 1320-1323). The problem which the present invention proposes to solve is to modify the region for interaction of the adenoviral fiber with the cellular receptor(s) in order to alter the natural host specificity of the adenoviruses carrying the mutated fiber. For ease of understanding, the term xe2x80x9ccellular receptorxe2x80x9d for adenoviruses will be used hereinafter to designate the cellular polypeptide(s) involved directly or otherwise in the binding of adenoviruses to their natural target cells or in the penetration into the latter. Of course, said receptor may be different depending on the serotypes. The addition of a ligand makes it possible to confer a new tropism toward one or more specific cell types carrying at their surface a target molecule recognized by the ligand in question.
The present invention constitutes an improvement of the previous technique since it discloses the regions of the fiber to be mutated in order to inhibit or prevent binding to the natural cellular receptor for adenoviruses. One or more residues of the 443 to 462 region of the head of the Ad5 fiber have now been substituted or deleted and an inhibition of the infectivity of the corresponding adenoviruses toward normally permissive cells has now been shown. The introduction of the GRP (for gastrin releasing peptide) ligand into these fibers should make it possible to target the infection toward cells expressing the GRP receptor. The aim of the present invention is to reduce the therapeutic quantities of adenoviruses to be used and to target the infection at the cells to be treated. This specificity is essential when an adenovirus expressing a cytotoxic gene is used, in order to avoid the propagation of the cytotoxic effect to healthy cells. The advantages offered by the present invention are to reduce the risks of dissemination and the secondary effects linked to the adenoviral technology.
Accordingly, the subject of the present invention is an adenovirus fiber modified by mutation of one or more residues of said fiber, characterized in that said residues are directed toward the natural cellular receptor for said adenovirus.
The term xe2x80x9cfiberxe2x80x9d is widely defined in the introductory part. The fiber of the present invention may be derived from an adenovirus of human, canine, avian, bovine, murine, ovine, porcine or simian origin or may be a hybrid and may comprise fragments of diverse origins. As regards human adenoviruses, the use of those of serotype C and, in particular, the type 2 or 5 adenoviruses (Ad2 or Ad5) is preferred. It is indicated that the Ad2 fiber contains 580 amino acids (aa) whose sequence is disclosed in Herissxc3xa9 et al. (1981, Nucleic Acid Res. 9, 4023-4042). That of Ad5 has 582 aa and its sequence, presented in the sequence identifier 1 (SEQ ID NO: 1) has been determined by Chroboczek and Jacrot (1987, Virology 161, 549-554). When the fiber of the present invention originates from an animal adenovirus, bovine adenoviruses and, in particular, those of the BAV-3 strain are preferably used. The latter have been the subject of numerous studies and the sequence of the fiber is disclosed in international application WO95/16048. Of course, the fiber of the present invention may exhibit other modifications compared to the native sequence, other than those which are the subject of the present invention.
In accordance with the aims pursued by the present invention, the fiber according to the invention is modified so as to reduce or abolish its capacity to bind to the natural cellular receptor. Such a property may be verified by studying the infectivity or the cellular binding of the corresponding viruses by applying the techniques of the art such as those detailed below. According to an advantageous embodiment, the trimerization and penton-base-binding properties are not affected.
For the purposes of the present invention, the term xe2x80x9cmutationxe2x80x9d designates a deletion, substitution or addition of one or more residues or a combination of these possibilities. The case where the regions for interaction with the natural cellular receptor are deleted completely or partly and replaced in particular with a ligand specific for a cell surface protein other than the natural receptor for adenoviruses is most particularly preferred.
The three-dimensional crystallographic structure of the adenoviral head has been determined by Xia et al. (1994, Structure 2, 1259-1270). Each monomer contains 8 antiparallel xcex2 sheets designated A to D and G to J and 6 major loops of 8 to 55 residues. For example, the CD loop links the xcex2 sheet C to the xcex2 sheet D. It is indicated that the minor sheets E and F are considered to be part of the DG loop situated between the D and G sheets. As a guide, Table 1 indicates the location of these structures in the amino acid sequence of the fiber of Ad5 as shown in the sequence identifier No. 1 (SEQ ID NO: 1), +1 representing the initiator Met residue. In general, the sheets form an ordered and compact structure whereas the loops are more flexible. These terms are conventional in the field of protein biochemistry and are defined in basic manuals (see for example Stryer, Biochemistry, 2nd edition, Chap 2, p 11 to 39, Ed. Freeman and Company, San Francisco).
The four xcex2 sheets A, B, C and J constitute the V sheets directed toward the viral particle. The other four (D, G, H and I) form the R sheets, which are supposed to face the cellular receptor. The V sheets appear to play an important role in the trimerization of the structure whereas the R sheets are thought to be involved in the interaction with the receptor. The residues of the fiber of Ad2, Ad3, Ad5, Ad7, Ad40, Ad41 and of the canine adenovirus CAV forming these different structures are clearly indicated in the preceding reference.
The modifications of the adenoviral fiber according to the invention affect more particularly the domain extending from the CD loop to the I sheet and involve in particular residues 441 to 557 of the Ad5 fiber and 441 to 558 of the Ad2 fiber. As a result of their spatial location in the native fiber, these residues are capable of recognizing and/or interacting directly or indirectly with the natural cellular receptor for the adenovirus in question. Inside this region, it is preferable to modify the part which comprises the CD loop, the D sheet and the proximal part of the DG loop (positions 441 to 478 of the Ad2 and Ad5 fiber) and, more particularly, the region extending from residues 443 to 462 as regards Ad5 or 451 to 466 in the case of Ad2. The other target region for the modifications is the H sheet (aa 529 to 536 of the Ad5 fiber and of the Ad2 fiber). Another alternative consists in the modification of the minor sheets E (aa 479-482 Ad5) and F (aa 485-486 Ad5).
As indicated above, it is possible to carry out the procedure by substituting one or more amino acids in the regions exposed. There may be mentioned in this regard the following examples which are derived from the Ad5 fiber in which:
the glycine residue at position 443 is substituted by an aspartic acid,
the leucine residue at position 445 is substituted by a phenylalanine,
the glycine residue at position 450 is substituted by an asparagine,
the threonine residue at position 451 is substituted by a lysine,
the valine residue at position 452 is substituted by an asparagine,
the alanine residue at position 455 is substituted by a phenylalanine,
the leucine residue at 457 is substituted by an alanine or a lysine, and/or
the isoleucine residue at position 459 is substituted by an alanine.
It is also possible to introduce several substitutions into the targeted region of the fiber, in particular at the level of the amino acids forming an elbow, preferably of the xcex1xcex1 type (see Table 2 by Xia et al., 1994, supra). By way of illustration, there may be mentioned the following two examples in which the Ad5 fiber is modified by substitution:
of the glycine residue at position 443 by an aspartic acid,
of the serine residue at position 444 by a lysine, and
of the alanine residue at position 446 by a threonine; or
of the serine residue at position 449 by an aspartic acid,
of the glycine residue at position 450 by a lysine,
of the threonine residue at position 451 by a leucine, and
of the valine residue at position 452 by a threonine.
Of course, the replacement amino acids are only mentioned as guide and any amino acid may be suitable for the purposes of the present invention. It is preferable, nevertheless, not to drastically modify the three-dimensional structure. Preferably, the amino acids forming an elbow will be replaced by residues forming a similar structure such as those described in the Xia et al. reference already mentioned.
The fiber of the present invention may also be modified by deletion. The region eliminated may involve all or part of the domain exposed and, in particular, of the CD loop, of the D sheet, of the DG loop and/or of the E and F sheets. As regards an Ad5 fiber according to the invention, there may be mentioned more particular the deletion:
of the region extending from the serine at position 454 to the phenylalanine at position 461,
of the region extending from the valine at position 441 to the glutamine at position 453,
of the region extending from the valine at position 441 to the phenylalanine at position 461, or
of the region extending from the asparagine at position 479 to the threonine at position 486.
It is also possible to generate other mutants from substitution or deletion in the other sheets or loops, such as for example the G, H and I sheets and the HI and DG loops.
According to an advantageous embodiment, when at least one of the modifications is a deletion of at least 3 consecutive residues of a loop and/or of a sheet, the deleted residues may be replaced by residues of an equivalent loop and/or sheet derived from a fiber of a second adenovirus capable of interacting with a cellular receptor different from that recognized by the first adenovirus. The second adenovirus may be of any origin, human or animal. This makes it possible to maintain the structure of the fiber according to the invention while conferring on it a host specificity corresponding to that of the second adenovirus. As indicated in Xia et al. (1994, supra), the cellular receptor mediating the infection by types 2 and 5 adenoviruses is different from that interacting with the types 3 and 7 adenoviruses. Thus, an Ad5 or Ad2 fiber deleted for at least 3 consecutive residues among those specified above may be substituted by the residues derived from an equivalent region of the Ad3 or Ad7 fiber in order to reduce its capacity to bind the Ad5 receptor and confer on it a new specificity toward the cellular receptor for Ad3 or Ad7. By way of nonlimiting example, there may be mentioned the replacement of the LAPISGTVQSAHLIITRFD (SEQ ID NO: 41) residues (positions 445 to 462) of the Ad5 fiber with the VNTLFKNKNVSINVELYFD (SEQ ID NO: 42) residues of the Ad3 fiber of the replacement of the PVTLTITL (SEQ ID NO: 43) residues (position 529 to 536) of the fiber of Ad5 with the PLEVTVML (SEQ ID NO: 44) residues of the fiber of Ad3.
The present invention also relates to an adenovirus fiber having a substantially reduced capacity for binding to the natural cellular receptor and nevertheless capable of trimerizing and of binding the penton base. As indicated above, the natural cellular receptor is advantageously chosen from the group consisting of the class I major histocompatibility antigens, fibronectin and the cellular receptor for the coxsackie viruses (CAR) or any other cell surface determinant which is usually involved or which participates in the infectivity of adenoviruses.
According to an equally advantageous embodiment, the fiber according to the invention comprises, in addition, a ligand. For the purposes of the present invention, the term ligand defines any entity capable of recognizing and binding, preferably with a high affinity, a cell surface molecule different from the natural cellular receptor. This molecule may be expressed or exposed at the surface of the cell which it is desired to target (cell surface marker, receptor, antigenic peptide presented by histocompatibility antigens and the like). In accordance with the aims pursued by the present invention, a ligand may be an antibody, a peptide, a hormone, a polypeptide or a sugar. The term antibody comprises in particular monoclonal antibodies, antibody fragments (Fab) and single-chain antibodies (scFv). These names and abbreviations are conventional in the field of immunology.
Within the framework of the present invention, it may be advantageous to target more particularly a tumor cell, an infected cell, a particular cell type or a category of cells carrying a specific surface marker. For example, if the host cell to be targeted is a cell infected with the HIV virus (Human Immunodeficiency Virus), the ligand may be a fragment of antibody against fusin, the CD4 receptor or against an exposed viral protein (envelope glycoprotein) or the part of the TAT protein of the HIV virus extending from residues 37 to 72; (Fawell et al., 1994, Proc. Natl. Acad. Sci. USA 91, 664-668). As regards a tumor cell, the choice will be on a ligand recognizing an antigen specific for tumors (for example the MUC-1 protein in the case of breast cancer, some epitopes of the E6 or E7 proteins of the papillomavirus HPV) or overexpressed (receptor for IL-2 overexpressed in some lymphoid tumors, GRP peptide, for Gastrin Releasing Peptide, overexpressed in lung carcinoma cells (Michael et al., 1995 Gene Therapy 2, 660-668) and in pancreas, prostate and stomach tumors). If it is desired to target the T lymphocytes, it is possible to use a ligand for the T cell receptor. Moreover, transferrin is a good candidate for hepatic screening. In general, the ligands which may be used in the context of the invention are widely described in the literature and may be cloned by standard techniques. It is also possible to synthesize them by the chemical route and to couple them to the fiber according to the invention. In this regard, the coupling of galactosyl residues should confer a hepatic specificity because of the interaction with the asialoglycoprotein receptors. However, the preferred embodiment consists in inserting the ligand at the C-terminal end of the fiber according to the invention or as a replacement for the deleted residues when at least one of the modifications is a deletion of at least 3 consecutive residues.
The present invention also relates to a DNA fragment encoding a fiber according to the invention as well as to a vector for expressing such a fragment. Any type of vector may be used to this effect, whether it is of plasmid or viral origin, integrative or otherwise. Such vectors are commercially available or are described in the literature. Likewise, persons skilled in the art are capable of adapting the regulatory elements necessary for the expression of the DNA fragment according to the invention. In addition, it may be combined with one or more substances capable of improving the transfection efficiency and/or the stability of the vector. These substances are widely documented in the literature accessible to persons skilled in the art (see for example Felgner et al., 1989, Proc. West. Pharmacol. Soc. 32, 115-121; Hodgson and Solaiman, 1996, Nature Biotechnology 14, 339-342; Remy et al., 1994, Bioconjugate chemistry 5, 647-654). By way of nonlimiting illustration, these may be polymers, lipids, in particular cationic lipids, liposomes, nuclear proteins and neutral lipids. A combination which can be envisaged is a vector combined with cationic lipids (DC-Chol, DOGS and the like) and neutral lipids (DOPE).
The present invention also relates to an adenovirus lacking a functional native fiber and which comprises, at its surface, a fiber according to the invention. The latter may be expressed by the adenoviral genome or provided in trans by a complementation cell line, such as those defined below. It may, in addition, comprise a ligand as defined above. Preferably, the specificity of binding of such an adenovirus to its natural cellular receptor is significantly reduced or, even better, abolished, because of the modified fiber which it carries. The loss of the natural specificity may be evaluated by studies of cellular attachment carried out in the presence of labeled viruses (for example labeled with 3H-thymidine according to the technique of Roelvink et al., 1996, J. Virol. 70, 7614-7621) or by studies of infectivity of cells which are permissive or which express the surface molecule targeted by the ligand (see the examples which follow).
The ligand may be chemically coupled to the adenovirus according to the invention. However, the variant according to which the sequences encoding the ligand are inserted into the adenoviral genome, in particular, into sequences encoding the modified fiber according to the invention, preferably, in phase in order to preserve the reading frame, is preferred. The insertion may take place at any site. However, the preferred site of insertion is upstream of the stop codon at the C-terminal end or in place of the deleted residues. It is also possible to envisage introducing the sequences of the ligand into other adenoviral sequences, in particular those encoding another capsid protein, such as the hexon or the penton.
Advantageously, an adenovirus according to the invention is recombinant and replication-defective, that is to say incapable of autonomously replicating in a host cell. The deficiency is obtained by mutation or deletion of one or more essential viral genes and, in particular, of all or part of the E1 region. Deletions in the E3 region may be envisaged in order to increase the cloning capacities. However, it may be advantageous to conserve the sequences encoding the gp19 k protein (Gooding and Wood, 1990, Critical Reviews of Immunology 10, 53-71) in order to modulate the immune responses of the host. Of course, the genome of an adenovirus according to the invention may also comprise additional deletions or mutations affecting other regions, in particular the E2, E4 and/or L1-L5 regions (see for example international application WO94/28152 and Ensinger et al., 1972, J. Virol. 10, 328-339 describing the heat-sensitive mutation of the DBP gene of E2).
According to a preferred embodiment, an adenovirus according to the invention is recombinant and comprises one or more genes of interest placed under the control of the elements necessary for their expression in a host cell. The gene in question may be of any origin, genomic, cDNA (complementary DNA) or hybrid (minigene lacking one or more introns). It may be obtained by conventional molecular biology techniques or by chemical synthesis. It may encode an antisense RNA, a ribozyme or an mRNA which will then be translated into a polypeptide of interest. The latter may be cytoplasmic, membranal or may be secreted from the host cell. Moreover, it may be all or part of a polypeptide as found in nature, a chimeric polypeptide obtained from the fusion of sequences of diverse origins, or of a polypeptide mutated relative to the native sequence having improved and/or modified biological properties.
In the context of the present invention, it may be advantageous to use the genes encoding the following polypeptides:
cytokines or lymphokines (xcex1-, xcex2- and xcex3-interferons, interleukins and in particular IL-2, IL-6, IL-10 or IL-12, tumor necrosis factors (TNF), colony stimulating factors (GM-CSF, C-CSF, M-CSF and the like);
cellular or nuclear receptors, in particular those recognized by pathogenic organisms (viruses, bacteria or parasites) and, preferably, by the HIV virus or their ligands (fas ligand);
proteins involved in a genetic disease (factor VII, factor VIII, factor IX, dystrophin or minidystrophin, insulin, CFTR protein (Cystic Fibrosis Transmembrane Conductance Regulator), growth hormones (hGH);
enzymes (urease, renin, thrombin and the like);
enzyme inhibitors (xcex11-antitrypsin, antithrombin III, viral protease inhibitors and the like);
polypeptides with antitumor effect which are capable of at least partially inhibiting the initiation or the progression of tumors or cancers (antibodies, inhibitors acting on cell division or transduction signals, products of expression of tumor suppressor genes, for example p53 or Rb, proteins stimulating the immune system and the like);
proteins of the class I or II major histocompatibility complex or regulatory proteins acting on the expression of the corresponding genes;
polypeptides capable of inhibiting a viral, bacterial or parasitic infection or its development (antigenic polypeptides having immunogenic properties, antigenic epitopes, antibodies, transdominant variants capable of inhibiting the action of a native protein by competition and the like);
toxins (herpes simplex virus 1 thymidine kinase (HSV-1-TK), ricin, cholera toxin, diphtheria toxin and the like) or immunotoxins,
markers (xcex2-galactosidase, luciferase and the like),
polypeptide having an effect on apoptosis (inducer of apoptosis: Bax and the like, inducer of apoptosis Bcl2, Bclx), cytostatic agents (p21, p16, Rb and the like), apolipoproteins (apoE and the like), SOD, catalase, nitric oxide synthase (NOS); and
growth factors (FGF for Fibroblast growth Factor, VEGF for Vascular Endothelial, cell growth Factor and the like).
It should be noted that this list is not limiting and that other genes may also be used.
Moreover, an adenovirus according to the invention may, in addition, comprise a selectable gene which makes it possible to select or identify the infected cells. There may be mentioned the genes neo (encoding neomycin phosphotransferase) conferring resistance to the antibiotic G418, dhfr (Dihydrofolate Reductase), CAT (Chloramphenicol Acetyl transferase), pac (Puromycin Acetyl-Transferase) or gpt (Xanthine Guanine Phosphoriboxyl Transferase). In general, the selectable genes are known to a person skilled in the art.
Elements necessary for the expression of a gene of interest in a host cell are understood to mean all the elements allowing its transcription into RNA and the translation of an mRNA into a protein. Among these, the promoter is of particular importance. In the context of the present invention, it may be derived from any gene of eukaryotic or even viral origin and may be constitutive or regulatable. Moreover, it may be modified so as to improve the promoter activity, suppress a transcription-inhibiting region, make a constitutive promoter regulatable or vice versa, introduce a restriction site and the like. Alternatively, it may be the natural promoter of the gene to be expressed. There may be mentioned, by way of examples, the CMV (Cytomegalovirus) viral promoter, the RSV (Rous Sarcoma Virus) viral promoter, the promoter of the HSV-1 virus TK gene, the early promoter of the SV40 virus (Simian Virus 40), the adenoviral MLP promoter or the eukaryotic promoters of the murine or human genes for PGK (Phospho Glycerate kinase), MT (metallothionein), xcex11-antitrypsin and albumin (liver-specific), immunoglobulins (lymphocyte-specific). It is also possible to use a tumor-specific promoter (xcex1-fetoprotein AFP, Ido et al., 1995, Cancer Res. 55, 3105-3109; MUC-1; PSA for prostate specific antigen, Lee et al., 1996, J. Biol. Chem. 271, 4561-4568; and flt1 specific for endothelial cells, Morishita et al., 1995, J. Biol. Chem. 270, 27948-27953).
Of course, a gene of interest in use in the present invention may, in addition, comprise additional elements necessary for the expression (intron sequence, signal sequence, nuclear localization sequence, transcription terminating sequence, site for initiation of translation of the IRES type and the like) or for its maintenance in the host cell. Such elements are known to persons skilled in the art.
The invention also relates to a method of preparing an adenovirus according to the invention, according to which
the genome of said adenovirus is transfected into an appropriate cell line,
said transfected cell line is cultured under appropriate conditions in order to allow the production of said adenovirus, and
said adenovirus is recovered from the culture of said transfected cell line and, optionally, said adenovirus is substantially purified.
The choice of the cell line depends on the deficient functions in the adenovirus according to the invention and a complementation line capable of providing in trans the deficient function(s) will be used. The 293 line is suitable for complementing the E1 function (Graham et al., 1977, J. Gen. Virol. 36, 59-72). For a double deficiency E1 and E2 or E4, it is possible to use a line among those described in French patent application 96 04413. It is also possible to use a helper virus to complement the defective adenovirus according to the invention in any host cell or a mixed system using complementation cell and helper virus in which the elements are dependent on each other. The means for propagating a defective adenovirus are known to a person skilled in the art who may refer, for example, to Graham and Prevec (1991, Methods in Molecular Biology, vol. 7, p. 190-128; Ed. E. J. Murey, The Human Press Inc.). The adenoviral genome is preferably reconstituted in vitro in Escherichia coli (E. coli) by ligation or by homologous recombination (see for example French application 94 14470). The methods of purification are described in the state of the art. There may be mentioned the density gradient centrifugation technique.
The present invention also relates to a cell line comprising, either in a form integrated into the genome or in the form of an episome, a DNA fragment encoding a fiber according to the invention, placed under the control of the elements allowing its expression. Said line may, in addition, be capable of complementing an adenovirus deficient for one or more functions selected from those encoded by the E1, E2, E4 and L1-L5 regions. It is preferably derived from the 293 line. Such a line may be useful for the preparation of an adenovirus whose genome lacks all or part of the sequences encoding the fiber (so as to produce a nonfunctional fiber). The subject of the present invention is also the corresponding method, according to which:
the genome of said adenovirus is transfected into a cell line according to the invention,
said transfected cell line is cultured under appropriate conditions in order to allow the production of said adenovirus, and
said adenovirus is recovered from the culture of said transfected cell line and, optionally, said adenovirus is substantially purified.
The present invention also covers a host cell infected with an adenovirus according to the invention or capable of being obtained by a method according to the invention. This is advantageously a mammalian cell and, in particular, a human cell. It may be a primary or tumor cell and of any origin, for example of hematopoietic origin (totipotent stem cell, leukocyte, lymphocyte, monocyte or macrophage and the like), muscle (satellite cell, myocyte, myoblast, smooth muscle cell), cardiac, nasal, pulmonary, tracheal, hepatic, epithelial or fibroblast origin.
The subject of the invention is also a pharmaceutical composition comprising, as therapeutic or prophylactic agent, a host cell or an adenovirus according to the invention or capable of being obtained by a method according to the invention, in combination with a pharmaceutically acceptable carrier. The composition according to the invention is, in particular, intended for the preventive or curative treatment of diseases such as genetic diseases (hemophilia, cystic fibrosis, diabetes, Duchenne""s. myopathy or Becker""s myopathy and the like), cancers, such as those induced by oncogenes or viruses, viral diseases, such as hepatitis B or C and AIDS (acquired immunodeficiency syndrome resulting from HIV infection), recurring viral diseases, such as viral infections caused by the herpesvirus and cardiovascular diseases including restenoses.
A pharmaceutical composition according to the invention may be manufactured conventionally. In particular, a therapeutically effective quantity of the therapeutic or prophylactic agent is combined with a carrier such as a diluent. A composition according to the invention may be administered by the local, systemic or aerosol route, in particular by the intragastric, subcutaneous, intracardiac, intra-muscular, intravenous, intraperitoneal, intratumor, intrapulmonary, intranasal or intracheal route. The administration may take place in a single dose or repeated once or several times after a certain time interval. The appropriate route of administration and the appropriate dosage vary according to various parameters, for example, the individual or patient to be treated or the gene(s) of interest to be transferred. In particular, the viral particles according to the invention may be formulated in the form of doses of between 104 and 1014 pfu (plaque-forming units), advantageously 105 and 1013 pfu and, preferably, 106 and 1012 pfu. The formulation may also include a diluent, an adjuvant, a pharmaceutically acceptable excipient as well as a stabilizer, preservative and/or solubilizer. A formulation in saline, nonaqueous or isotonic solution is particularly suitable for an injectable administration. It may be provided in liquid or dry form (for example a lyophilisate and the like) or any other galenic form commonly used in the pharmaceutical field.
Finally, the present invention relates to the therapeutic or prophylactic use of an adenovirus or of a host cell according to the invention or of an adenovirus capable of being obtained by a method according to the invention, for the preparation of a medicament intended for the treatment of the human or animal body by gene therapy. According to a first possibility, the medicament may be administered directly in vivo (for example by intravenous injection, into an accessible tumor, into the lungs by aerosol and the like). It is also possible to adopt the ex vivo approach which consists in collecting cells from the patient (bone marrow stem cells, peripheral blood lymphocytes, muscle cells and the like), transfecting or infecting them in vitro according to prior art techniques and readministering them to the patient.
The invention also extends to a method of treatment according to which a therapeutically effective quantity of an adenovirus or of a host cell according to the invention is administered to a patient requiring such a treatment.